The pathophysiology of preeclampsia (PE) is unclear, and effective strategies/treatments to successfully reduce global maternal and neonatal morbidity/mortality are lacking. The long term objective of this project is to improve our understanding of the biologic underpinnings of PE so that effective prevention, detection, and treatment interventions can be developed to identify women at risk and improve health outcomes of moms and babies affected by PE. This project aims to address significant gaps in our understanding of PE pathophysiology by focusing on improving our understanding of endoglin (ENG) pathway dysregulation in PE. We know that elevations in circulating soluble endoglin (sENG) protein and placental/blood ENG mRNA expression precede clinical presentation of PE, and increased levels of sENG in rodents produce a PE-like syndrome that can be amplified by other anti-angiogenic factors. I have previously demonstrated that genetic variation in the ENG pathway is associated with PE in American Caucasian and African American samples. However, it is unknown if genetic variability in the ENG pathway impacts ENG mRNA expression and/or sENG levels, or if metabolic factors (e.g., Vitamin D down in PE) modulate cellular sENG output. This project addresses mechanisms of endoglin pathway dysregulation and validation of our previous genetic findings, both of which are necessary to provide the evidence based needed to eventually translate these findings clinically. First, using a candidate gene case-control design, we will test the hypothesis that our genetic association findings are present in other populations from the Bill & Melinda Gates funded Global Pregnancy CoLaboratory, a biobank consortium of >20 research groups studying adverse pregnancy outcomes. Second, we will conduct focused genotyping (missense single nucleotide polymorphism assessment; sequencing) of ENG to identify common and/or rare functional variants that are involved in PE susceptibility, using a case-control design. Third, we will explor mechanisms underlying associations between the ENG pathway: (a) test the relationship of pregnancy plasma levels (e.g., sENG) to ENG pathway genotypes (within-case OR within-control design) and pregnancy outcome (case-control design); (b) test the hypothesis that Vitamin D attenuates sENG release from human trophoblast and uterine endothelial cell lines cultured under basal and stimulated (hypoxia, PE serum) conditions. Methods to achieve these specific aims, germane to the National Institute of Nursing Research's mission to promote health and prevent disease through identification of susceptibility genes for at- risk individuals, includ genotype collection with either the i-PLEX(R) Gold SNP assay or TaqMan(R) allelic discrimination, capillary based sequencing, protein measurement with ELISA assays and Western Blot, and cell culture under varying treatment conditions (e.g., oxygen content, Vitamin D concentration, patient serum). Ultimately, results from this project will improve our understanding of the pathophysiologic underpinnings of PE and may help to identify preventive, predictive, and therapeutic/modifiable targets for women at risk for PE.